2-h-indazole derivatives as cyclin-dependent kinase (cdk) inhibotors and therapeutic uses thereof

ABSTRACT

Indazole compounds of formula (I) as cyclin-dependent kinase (CDK) and cell-proliferation inhibitors, and therapeutic uses and methods of preparation thereof, are disclosed. These compounds, and pharmaceutically acceptable salts, solvates, prodrugs, and pharmaceutical compositions thereof, are useful for treating diseases and disorders associated with activity of cyclin-dependent kinases, in particular CDK4/6, including but not limited to various cancers and inflammation-related diseases or conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/328,813, filed on Jan. 24, 2017, which is the U.S. National Phase ofInternational Application No. PCT/US2015/041915, filed on Jul. 24, 2015,which in turn claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/028,427, filed on Jul. 24, 2014, thedisclosures of all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention is related to the field of compounds, compositionsand methods for the treatment or prevention of a disease, disorder, ormedical condition mediated through certain cyclin-dependent kinases(CDKs). The diseases include various cancers.

BACKGROUND OF THE INVENTION

Cyclin-dependent kinases are a family of protein kinases that regulatecell division and proliferation. Cell cycle progression is controlled bycyclins and their associated cyclin-dependent kinases, such as CDK1,CDK2, CDK3, CDK4 and CDK6, while other CDKs such as CDK7, CDK8 and CDK9are critical to transcription. CDK binding to cyclins formsheterodimeric complexes that phosphorylate their substrates on serineand threonine residues, which in turn initiates events required forcell-cycle transcription and progression. Since uncontrolled cellproliferation is a hallmark of cancer, and most cancer cells exhibitderegulation of CDKs, inhibition of CDKs has emerged as a potentialtreatment for various cancers. Inhibitors with varying degrees ofselectivity for CDKs have been reported; however, selective CDK4/6inhibitors are currently viewed as a promising class of potentialanticancer or anti-inflammatory agents due to both the critical role ofCDK4/6 in regulating cell proliferation and the toxic effects associatedwith inhibition of other members of the CDK family.

Recently, several types of aminopyrimidine derivatives have beenreported to be selective CDK4/6 inhibitors. See, e.g., WO 2003/062236,WO 2007/140222, and US 2010/0160340. Each of these types of moleculescontains a 2-aminopyrimidine moiety bound through the 2-amino group toan aryl or heteroaryl ring system. There remains a need to develop newCDK 4/6 inhibitors as novel anticancer and/or anti-inflammatory agents.

SUMMARY OF THE INVENTION

The present invention relates to 2-aminopyrimidine-substituted indazolederivatives that are effective as selective CDK inhibitors and useful inthe treatment or prevention of diseases, disorders, or medicalconditions mediated through certain CDKs, in particular CDK4 and CDK6,such as various types of cancers and inflammation-related conditions.

One aspect of the present invention is directed to a compound of formula(I):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

R¹ is selected from the group consisting of hydrogen, linear or branchedC₁-C₈ alkyl, and C₃-C₇ cycloalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, linear or branched C₁-C₈ alkyl, C₃-C₇ cycloalkyl, andcycloalkylmethyl;

R⁴ is selected from the group consisting of hydrogen, halogen, linear orbranched C₁-C₈ alkyl, and C₃-C₇ cycloalkyl; and

R⁵ is hydrogen or halogen.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, and one or morepharmaceutically acceptable excipients, such as adjuvants, diluents,and/or carriers.

Another aspect of the present invention is directed to a method oftreating a disease, disorder, or condition mediated through at least oneof cyclin-dependent kinases (CDK), in particular CDK4, CDK6, or acombination thereof, comprising administering to a subject in needthereof a therapeutically effective amount of a compound of formula (I),or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Another aspect of the present invention is directed to a method oftreating a disease, disorder, or condition mediated through at least oneof cyclin-dependent kinases (CDK), in particular CDK4, CDK6, or acombination thereof, comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, and one or morepharmaceutically acceptable excipients, such as adjuvants, diluents,and/or carriers.

In one embodiment, the diseases, disorders, or conditions associatedwith one or more cyclin-dependent kinases, in particular CDK4, CDK6, ora combination thereof, comprise cancers, which may include, but are notlimited to, lung cancer, especially non-small cell lung cancer (NSCLC),breast cancer, prostate cancer, colorectal cancer, glioblastoma, mantelcell lymphoma, chronic myeloid leukemia and acute myeloid leukemia, andcomplications thereof.

In another embodiment, the diseases, disorders, or conditions comprisethe inflammation-related diseases and conditions, such as arthritis,e.g., rheumatic arthritis, and cystic fibrosis.

Another aspect of the invention is directed to a method of inhibitingcell proliferation comprising treating the cells with an effectiveamount of a compound of formula (I), or a salt, solvate, prodrug, orcomposition thereof.

Another aspect of the invention is directed to a method of inhibiting acyclin-dependent kinase (CDK), in particular CDK4, CDK6, or acombination thereof, comprising treating the kinase with an effectiveamount of a compound of formula (I), or a salt, solvate, prodrug, orcomposition thereof.

Another aspect of the present invention is directed to use of thecompounds of this invention for the study of CDKs in biological andpathological phenomena and for comparative evaluation of new kinaseinhibitors.

Another aspect of the present invention is directed to use of a compoundof formula (I) according to any embodiments described herein, or apharmaceutically acceptable salt, solvate, prodrug, or compositionthereof, in the manufacture of a medicament for treatment of a diseaseor disorder associated with a CDK activity. The CDK activity ispreferably activity of CDK4, CDK6, or a combination thereof.

Still another aspect of the present invention is directed to the methodsof synthesizing the compounds of formula (I) as substantially disclosedand described herein.

Other aspects or advantages of the present invention will be apparent tothose skilled in the art in view of the following detailed descriptionand claims in combination with the knowledge and skills generally knownin the field.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel 2-aminopyrimidine-substituted2H-indazole derivatives useful as CDK inhibitors.

In one aspect, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

R¹ is selected from the group consisting of hydrogen, C₁-C₈ alkyl, andC₃-C₇ cycloalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, linear or branched C₁-C₈ alkyl, C₃-C₇ cycloalkyl, and C₃-C₇cycloalkylmethyl;

R⁴ is selected from the group consisting of hydrogen, halogen, linear orbranched C₁-C₈ alkyl, and C₃-C₇ cycloalkyl; and

R⁵ is hydrogen or halogen.

In one embodiment of this aspect, R¹ is C₁-C₆ alkyl.

In another embodiment of this aspect, R¹ is methyl, ethyl, propyl, orisopropyl.

In another embodiment of this aspect, R² is C₁-C₆ alkyl, C₃-C₆cycloalkyl, or C₃-C₆ cycloalkylmethyl.

In another embodiment of this aspect, R² is methyl, ethyl, propyl,isopropyl, cyclopropyl, cyclopentyl, cyclopropylmethyl, orcyclopentylmethyl.

In another embodiment of this aspect, R³ is C₁-C₆ alkyl or C₃-C₆cycloalkyl.

In another embodiment of this aspect, R³ is methyl, ethyl, propyl,isopropyl, or cyclopropyl.

In certain embodiments, the present invention provides a compound offormula (I), wherein the R⁴ substituent is attached at the 7-position ofthe indazole moiety, as in formula (Ia):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

R¹ is selected from the group consisting of hydrogen, C₁-C₈ alkyl, andC₃-C₇ cycloalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl, C₃-C₇ cycloalkyl, and cycloalkylmethyl;

R⁴ is selected from the group consisting of hydrogen, halogen, C₁-C₈alkyl, and C₃-C₇ cycloalkyl; and

R⁵ is hydrogen or halogen.

In another embodiment of this aspect, R⁴ is hydrogen or halogen.

In another embodiment of this aspect, R⁵ is hydrogen or fluoride.

In another embodiment of this aspect, R¹ is methyl or ethyl; R² isisopropyl, cyclopropyl, cyclopropylmethyl, or cyclopentyl; R³ is methylor ethyl, R⁴ is hydrogen or fluoro, and R⁵ is hydrogen or fluoro.

In certain preferred embodiments of this aspect, the R⁴ substituent isattached at the 7-position of the indazole moiety, and R⁵ is fluorine,characterized by formula (Ib):

wherein R⁴ is preferably hydrogen or halogen; and when R⁴ is a halogen,it is preferably chlorine or fluorine, more preferably fluorine.

In certain preferred embodiments of this aspect, the present inventionprovides a compound of formula selected from the group consisting of:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein R¹, R², and R³ are each defined in any of the embodimentsdescribed here.

In certain preferred embodiments of this aspect, the present inventionprovides the compounds listed in Table 1 (infra), and pharmaceuticallyacceptable salts, solvates, and prodrugs thereof.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I), (Ia), (Ib), (Ic),(Id), or (Ie) according to any embodiments described here, or apharmaceutically acceptable salt, solvate, or prodrug thereof, and oneor more pharmaceutically acceptable adjuvants, diluents, and/orcarriers.

In another aspect, the present invention provides a method of treating adisease, disorder, or condition mediated through activity of at leastone cyclin-dependent kinase (CDK), comprising administering to a subjectin need thereof a therapeutically effective amount of the compound offormula (I), (Ia), (Ib), (Ic), (Id), or (Ie) according to any of theembodiments described herein, or a pharmaceutically acceptable salt,solvate, or prodrug thereof.

In one embodiment of this aspect, the present invention provides amethod of treating a disease, disorder, or condition mediated throughactivity of at least one cyclin-dependent kinase (CDK), comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a therapeutically effective amount of the compound of formula(I), (Ia), (Ib), (Ic), (Id), or (Ie) according to any of the embodimentsdescribed herein, or a pharmaceutically acceptable salt, solvate, orprodrug thereof, and one or more pharmaceutically acceptable adjuvants,diluents, and/or carriers.

In one preferred embodiment of this aspect, the at least one CDK isCDK4, CDK6, or a combination thereof.

In another preferred embodiment of this aspect, the disease or disorderis a cancer or an inflammation-related disease or condition.

In another preferred embodiment of this aspect, the inflammation-relateddisease or condition is arthritis, such as rheumatic arthritis, orcystic fibrosis.

In another preferred embodiment of this aspect, the cancer is selectedfrom, but not limited to, colorectal cancer, breast cancer, lung cancer,especially non-small cell lung cancer (NSCLC), prostate cancer,glioblastoma, mantel cell lymphoma (MCL), chronic myeloid leukemia(CML), acute myeloid leukemia (AML), and complications thereof.

In another embodiment of this aspect, the compound of the presentinvention may be administered to a subject in need thereof incombination with administration of a second therapeutic agent.

In another embodiment, the second therapeutic agent is a different CDKinhibitor, a human epidermal growth factor receptor (e.g., HER2)inhibitor, a serine/threonine kinase inhibitor, such as a mammaliantarget of rapamycin (mTOR) inhibitor, or an epidermal growth factorreceptor (EGFR) inhibitor.

In another aspect, the present invention provides a method of inhibitingcell proliferation, comprising treating the cells with an effectiveamount of the compound of formula (I) according to any of theembodiments described, or a salt, solvate, prodrug, or compositionthereof. The method of inhibiting cell proliferation can take place invivo, e.g., inside the body of a subject, or in vitro, e.g., in abiological sample containing the proliferative cells of a subject.

In a preferred embodiment of this aspect, the proliferative cells arecancer cells, such as, but not limited to, cells of colorectal cancer,breast cancer, lung cancer, especially non-small cell lung cancer(NSCLC), prostate cancer, glioblastoma, mantel cell lymphoma (MCL),chronic myeloid leukemia (CML), acute myeloid leukemia (AML), orcomplications thereof.

In another aspect, the present invention provides a method of inhibitinga cyclin-dependent kinase (CDK) comprising treating said kinase with aneffective amount of a compound of formula (I) according to anyembodiments described herein, or a salt, solvate, prodrug, orcomposition thereof. The method of inhibiting CDK can take place invivo, e.g., inside the body of a subject, or in vitro, e.g., in abiological sample containing the proliferative cells of a subject.

In a preferred embodiment of this aspect, the cyclin-dependent kinase isCDK4, CDK6, or a combination thereof.

In another aspect, the present invention provides use of a compound offormula (I), (Ia), (Ib), (Ic), (Id), or (Ie) according to anyembodiments described herein, or a pharmaceutically acceptable salt,solvate, prodrug, or composition thereof, in the manufacture of amedicament for treatment of a disease or disorder associated with a CDKactivity. The CDK activity is preferably activity of CDK4, CDK6, or acombination thereof.

In one embodiment of this aspect, the disease or disorder is selectedfrom the group consisting of colorectal cancer, breast cancer, lungcancer, especially non-small cell lung cancer (NSCLC), prostate cancer,glioblastoma, mantel cell lymphoma (MCL), chronic myeloid leukemia(CML), and acute myeloid leukemia (AML).

In another embodiment of this aspect, the disease or disorder is aninflammation-related disease or condition, such as arthritis, inparticular rheumatic arthritis, or cystic fibrosis.

In another aspect, the present invention provides a method of preparinga compound of formula (I), comprising a step of coupling intermediate Ewith intermediate G:

wherein R¹ through R⁵ are defined according to any of the embodimentsdescribed herein, and X³ is Cl, Br, or I.

In one embodiment of this aspect, the method further includes the stepsof converting intermediate C to intermediate D and coupling theintermediate D with a pyrimidine compound H to form the intermediate E:

wherein R^(x) and R^(y) are independent alkyl, aryl, cycloalkyl, oralternatively together form an alkylene group, each optionallysubstituted by one or more substituents independently selected fromC₁-C₄ alkyl, halogen or phenyl; and wherein X¹, X², and X³ are eachindependently Cl, Br, or I, on condition that the intermediate D coupleswith the compound H selectively at the X² site over the X³ site,preferably having a higher than 90:10 selectivity, more preferablyhaving a 95:5 selectivity, and most preferably exclusively at the X²site.

In one embodiment of this aspect, the method further includes the stepsof converting starting material S1 to intermediate A and converting theintermediate A to the intermediate C:

wherein X¹ is Cl, Br, I, or MeSO₃—; and wherein R² and R³ are as definedaccording to any of the embodiments described herein.

In another embodiment of this aspect, the method further includesconverting the intermediate A to the intermediate C, alternatively,comprises converting the intermediate A to an alcohol intermediate Bfollowed by reduction of the alcohol intermediate B to form theintermediate C:

wherein R^(2a) and R^(2b) are each independently hydrogen, alkyl,cycloalkyl, or together form an alkylene group so that the group

formed in the intermediate C is R² as defined according to any of theembodiments described herein.

In one embodiment of this aspect, the method further includes a step offorming the intermediate G through coupling the pyridine aldehydecompound S2 and the piperazine compound S3 to form an intermediate F,followed by converting the intermediate F to the intermediate G:

wherein X⁴ is selected from the group consisting of Cl, Br, I, and —NO₂;and

wherein said converting the intermediate F to the intermediate Gcomprises replacing X⁴ with NH₂ when X⁴ is Cl, Br, or I; oralternatively reducing the nitro group (—NO₂) to amino group (—NH₂) whenX⁴ is —NO₂.

Compounds provided by this invention are also useful for the study ofkinases in biological and pathological phenomena, the study oftransduction pathways mediated by such kinases, and the comparativeevaluation of new kinase inhibitors.

Unless otherwise indicated, the term “alkyl,” as used herein, isintended to include both branched and straight-chain saturated aliphatichydrocarbon groups containing 1 to 8 carbons, preferably 1 to 6, morepreferably 1 to 4, carbons. The term encompasses, but is not limited to,methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl,hexyl, or the like.

Unless otherwise indicated, the term “alkylene,” as used herein, refersto a bivalent saturated aliphatic radical derived from an alkane byremoval of two hydrogen atoms. Examples include, but are not limited to,methylene (—CH₂—), ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), or thelike.

Unless otherwise indicated, the term “cycloalkyl”, as used herein aloneor as a part of another group, includes saturated cyclic hydrocarbonradical having 3 to 8 carbons forming the ring. Examples include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

Unless otherwise indicated, the term “aryl”, as used herein alone or aspart of another group, refers to monocyclic or bicyclic aromatic radicalcontaining 6 to 10 carbons in the ring portion (such as phenyl andnaphthyl, including 1-naphthyl and 2-naphthyl).

“Halo” or “halogen” as used herein, refers to fluoro (F), chloro (Cl),bromo (Br), and iodo (I).

Further, the alkyl, alkylene, cycloalkyl, and cycloalkylmethyl groupsoptionally can be independently further substituted with one or more,preferably 1 to 3, substituents independently selected from the groupconsisting of halogen and C₁-C₄ alkyl.

The compounds of the present invention are generally recognized asorganic bases, which are able to react with acids, specificallypharmaceutically acceptable acids, to form pharmaceutically acceptablesalts.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. See, e.g., S. M. Berge etal., J. Pharm. Sci., 1977, 66, 1-19, which is incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Preferred pharmaceutically acceptable salts include thehydrochloride salts.

The term “solvate,” as used herein, means a physical association of acompound of this invention with a stoichiometric or non-stoichiometricamount of solvent molecules. For example, one molecule of the compoundassociates with one or more, preferably one to three, solvent molecules.It is also possible that multiple (e.g., 1.5 or 2) molecules of thecompound share one solvent molecule. This physical association mayinclude hydrogen bonding. In certain instances the solvates will becapable of isolation as crystalline solid. The solvent molecules in thesolvate may be present in a regular arrangement and/or a non-orderedarrangement. Exemplary solvates include, but are not limited to,hydrates, ethanolates, methanolates, and isopropanolates. Methods ofsolvation are generally known in the art.

The term “prodrug,” as used herein, refers to a derivative of a compoundthat can be transformed in vivo to yield the parent compound, forexample, by hydrolysis in blood. Common examples of prodrugs in thepresent invention include, but are not limited to, amide orphosphoramide forms of an active amine compound, for example, thecompound of formula (II):

wherein R⁶ is an acyl group (e.g., acetyl, propionyl, formyl, etc.) orphosphoryl [e.g., —P(═O)(OH)₂] group; or alternatively, when R¹ or R³ inan active compound is hydrogen, the corresponding amide or phosphoramidecompounds may serve as prodrugs. Such amide or phosphoramide prodrugcompounds may be prepared according to conventional methods as known inthe art.

When it is possible that, for use in therapy, therapeutically effectiveamounts of a compound of the present invention, or pharmaceuticallyacceptable salts or solvates thereof, may be administered as the rawchemical, it is possible to present the active ingredient as apharmaceutical composition. Accordingly, the disclosure further providespharmaceutical compositions, which include any compounds of the presentinvention, or pharmaceutically acceptable salts or solvates thereof, andone or more, preferably one to three, pharmaceutically acceptablecarriers, diluents, or other excipients. The carrier(s), diluent(s), orother excipient(s) must be acceptable in the sense of being compatiblewith the other ingredients of the formulation and not deleterious to thesubject being treated.

The term “pharmaceutically acceptable,” as used herein, refers to theproperty of those compounds, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of patients without excessivetoxicity, irritation, allergic response, or other problem orcomplication commensurate with a reasonable benefit/risk ratio, and areeffective for their intended use.

Pharmaceutical formulations may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Typically, the pharmaceutical compositions of this disclosure will beadministered from once every 1 to 5 days to about 1-5 times per day, oralternatively, as a continuous infusion. Such administration can be usedas a chronic or acute therapy. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage formwill vary depending on the condition being treated, the severity of thecondition, the time of administration, the route of administration, therate of excretion of the compound employed, the duration of treatment,and the age, gender, weight, and condition of the patient. Preferredunit dosage formulations are those containing a daily dose or sub-dose,as herein above recited, or an appropriate fraction thereof, of anactive ingredient. Generally, treatment is initiated with small dosagessubstantially less than the optimum dose of the compound. Thereafter,the dosage is increased by small increments until the optimum effectunder the circumstances is reached. In general, the compound is mostdesirably administered at a concentration level that will generallyafford effective results without causing substantial harmful ordeleterious side effects.

When the compositions of this disclosure comprise a combination of acompound of the present disclosure and one or more, preferably one ortwo, additional therapeutic or prophylactic agent, both the compound andthe additional agent are usually present at dosage levels of betweenabout 10 to 150%, and more preferably between about 10 and 80% of thedosage normally administered in a monotherapy regimen.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example, by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual, ortransdermal), vaginal, or parenteral (including subcutaneous,intracutaneous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intralesional, intravenous, or intradermalinjections or infusions) route. Such formulations may be prepared by anymethod known in the art of pharmacy, for example by bringing intoassociation the active ingredient with the carrier(s) or excipient(s).Oral administration or administration by injection are preferred.

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilemulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water, and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing, and coloringagent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate, or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate, or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents, and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, and the like. Lubricantsused in these dosage forms include sodium oleate, sodium chloride, andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, betonite, xanthan gum, and the like. Tablets areformulated, for example, by preparing a powder mixture, granulating orslugging, adding a lubricant and disintegrant, and pressing intotablets. A powder mixture is prepared by mixing the compound, suitablecomminuted, with a diluent or base as described above, and optionally,with a binder such as carboxymethylcellulose, an aliginate, gelating, orpolyvinyl pyrrolidone, a solution retardant such as paraffin, aresorption accelerator such as a quaternary salt and/or and absorptionagent such as betonite, kaolin, or dicalcium phosphate. The powdermixture can be granulated by wetting with a binder such as syrup, starchpaste, acadia mucilage, or solutions of cellulosic or polymericmaterials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc, ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present disclosure can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material, and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups, and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic vehicle. Solubilizers andemulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylenesorbitol ethers, preservatives, flavor additive such as peppermint oilor natural sweeteners, or saccharin or other artificial sweeteners, andthe like can also be added.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release, for example, by coating or embedding particulatematerial in polymers, wax, or the like.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavoringagents.

The term “subject” or “patient” includes both humans and other mammaliananimals, preferably humans.

The term “therapeutically effective amount” refers to an amount of acompound or composition that, when administered to a subject fortreating a disease, is sufficient to effect such treatment for thedisease. A “therapeutically effective amount” can vary depending on,inter alia, the compound, the disease and its severity, and the age,weight, or other factors of the subject to be treated. When applied toan individual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially, orsimultaneously.

In some embodiments, the term “treating” or “treatment” refers to: (i)inhibiting the disease, disorder, or condition, i.e., arresting itsdevelopment; (ii) relieving the disease, disorder, or condition, i.e.,causing regression of the disease, disorder, and/or condition; or (iii)preventing a disease, disorder or condition from occurring in a subjectthat may be predisposed to the disease, disorder, and/or condition buthas not yet been diagnosed as having it. Thus, in some embodiments,“treating” or “treatment” refers to ameliorating a disease or disorder,which may include ameliorating one or more physical parameters, thoughmaybe indiscernible by the subject being treated. In some embodiments,“treating” or “treatment” includes modulating the disease or disorder,either physically (e.g., stabilization of a discernible symptom) orphysiologically (e.g., stabilization of a physical parameter) or both.In yet some embodiments, “treating” or “treatment” includes delaying theonset of the disease or disorder.

METHODS Abbreviations

The following abbreviations may be used in this application:

B₂pin₂=bis(pinacolato)diboron

MeOH=methanol

LDA=lithium diisopropylamide

LiHMDS=lithium bis(trimethylsilyl)amide [LiN(SiMe3)2]

Pd(dppf)Cl₂=[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)

Pd₂(dba)₃=tris(dibenzylideneacetone)dipalladium(0)

Xantphos=4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

nBu₃P=tri-n-butylphosphine

DCM=dichloromethane

THF=tetrahydrofuran;

DIEA=DIPEA=diisopropylethylamine;

sat.=saturated aqueous solution;

aq.=aqueous

FCC=flash column chromatography using silica;

TFA=trifluoroacetic acid;

r.t.=room temperature;

DMF=N,N-dimethylformamide;

DMSO=dimethylsulfoxide;

DMA=N,N-dimethylacetamide;

EtOAc=ethyl acetate;

h=hour(s).

Chemical Synthesis Synthesis of Compounds of Formula (I)

The synthesis of compounds of formula (I) is exemplified in the GeneralSynthetic Schemes 1-4:

1. Synthesis of the Indazole Intermediate C (Scheme 1)

A suitable 5-halo-2-nitrobenzaldehyde starting material S1 (X¹=Cl, Br,or I) is allowed to react with a primary amine (R³NH₂) in the presenceof a phosphine, e.g., tri-butylphosphine, to form the indazolederivative A (Genung, N. E. et al. Org. Lett. 2014 16, 3114-3117), whichin turn is deprotonated at the 3-position using a strong base, e.g.,LDA, followed by reaction with an alkylation reagent R²X (X=e.g., Cl,Br, I, or methanesulfonate) to form the intermediate C with the desiredR², R³, and R⁴ in place. Alternatively, the deprotonated compound A canbe allowed to react with an aldehyde or ketone to form an alcoholadduct, which is reduced (e.g., by a dialkylsilane) to form the desiredintermediate C.

2. Synthesis of the Pyrimidine-Substituted Indazole Intermediate E(Scheme 2)

The intermediate C is allowed to undergo a boronation reaction in thepresence of a catalyst (e.g., a palladium catalyst) to form the boronateintermediate D, which is allowed to couple with a halogen-substitutedpyrimidine derivative H to form a 5-(pyrimidin-3-yl)-indazoleintermediate E.

3. Synthesis of the 2-Amino-5-Piperazinylmethyl-Pyridine Intermediate G(Scheme 3)

A 6-halogen or 6-nitro substituted pyridine-3-carbaldehyde startingmaterial S2 and a 1-R¹-substituted piperazine starting material S3 areallowed to undergo a reductive amination reaction to form a2-amino-5-piperazinylmethyl-pyridine intermediate F, which is in turnconverted to the 2-amino-5-piperazinylmethyl-pyridine intermediate Gthrough substitution of the halogen or reduction of nitro group on thepyridine ring.

4. Synthesis of Compounds of Formula (I) (Scheme 4)

Coupling of the pyrimidine-substituted indazole intermediate E with the2-amino-5-piperazinemethyl-pyridine intermediate G in the presence of acatalyst (e.g., palladium catalyst) provides the compound of formula(I).

Examples

The following non-limiting Examples further illustrate certain aspectsof the present invention. These compounds are prepared according to thegeneral Synthetic Schemes described above.

Example 1.N-(5-((4-Ethylpiperazin-yl)methyl)pyridin-2-yl-5-fluoro-4-(3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-amine

Synthesis of Compound 1

As an illustrated example, the synthesis of intermediates E,intermediate G, and Compound 1 are depicted in Schemes 5-7,respectively. In the following reaction schemes some specific reagentsor reaction conditions are provided solely for better understanding, butsuch specific reagents or conditions are not intended to be limitingwhatsoever. As a person of skill in the art would appreciate, anyspecific step of the reaction scheme could be accomplished using avariety of equivalent conditions in various aspects, such as reagents,temperature, catalysts, and/or solvents, etc.

Intermediate A

Into a 500-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen were placed 5-bromo-2-nitrobenzaldehyde (30.0 g,130.4 mmol, 1.0 equiv.), methanamine (71.5 mL, 1.1 equiv.), andpropan-2-ol (300 mL). The resulting solution was stirred for 4 h at 80°C. The mixture was cooled to rt and tributylphosphine (98 mL, 3.0equiv.) was added. The resulting solution was stirred for 12 h at 80° C.and then extracted with 500 mL of ethyl acetate. The resulting mixturewas washed sequentially with 300 mL of NH₄Cl (aq.) and 300 mL of brine.The mixture was dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column andeluted with ethyl acetate/petroleum ether (1:4). This resulted in 32 g(crude) of 5-bromo-2-methyl-2H-indazole A as a red oil: MS m/z MH⁺=211

Intermediate B

Into a 1 L 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen were placed 5-bromo-2-methyl-2H-indazole A(32.0 g, 128.9 mmol, 1.0 equiv., 85%) in tetrahydrofuran (300 mL). Tothe solution was added LDA (97.5 mL, 1.5 equiv., 2 M) at −78° C. Thesolution was stirred at 0-5° C. for 10 min, then cooled to −78° C. Tothe solution was added propan-2-one (11.3 g, 194.7 mmol, 1.5 equiv.).The resulting solution was stirred for 12 h at 25° C. The reaction wasthen quenched with 100 mL of aqueous sodium bicarbonate. The resultingsolution was extracted with 3×500 mL of ethyl acetate and the organiclayers combined, dried with anhydrous Na₂SO₄, and filtered. The filtratewas concentrated under vacuum, and the residue was applied onto a silicagel column and eluted with ethyl acetate/petroleum ether (1:10 to 1:2).This resulted in 20 g (58%) of2-(5-bromo-2-methyl-2H-indazol-3-yl)propan-2-ol B as a yellow oil: MSm/z M⁺=269

Intermediate C

Into a 500-mL round-bottom flask were placed2-(5-bromo-2-methyl-2H-indazol-3-yl)propan-2-ol B (20.0 g, 74.3 mmol,1.0 equiv.), triethylsilane (86.6 g, 744.4 mmol, 10.0 equiv.),trifluoroacetic acid (85.0 g, 752.0 mmol, 10.0 equiv.), anddichloromethane (200 mL). The resulting solution was stirred for 12 h at25° C. The resulting mixture was concentrated under vacuum. The solutionwas adjusted to pH 8 with sodium bicarbonate (aq, 2 M). The resultingsolution was extracted with 3×100 mL of ethyl acetate, and the organiclayers combined and washed with 1×100 mL of brine. The mixture was driedwith anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue was applied onto a silica gel column and eluted with ethylacetate/petroleum ether (1:20 to 1:10). This resulted in 8 g (43%) of5-bromo-2-methyl-3-(propan-2-yl)-2H-indazole C as a yellow oil: MS[M+1]⁺=253 & 255 and [M+CH₃CN+H]⁺=294 &296

Intermediate D

Into a 100 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen were placed5-bromo-2-methyl-3-(propan-2-yl)-2H-indazole (1.9 g, 7.5 mmol, 1.0equiv.),4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane(2.3 g, 9.0 mmol, 1.2 equiv.), KOAc (1.47 g, 15.0 mmol, 2.0 equiv.),1,4-dioxane (40 mL), and water (10 mL). To the solution was addedPd(dppf)Cl₂ (612 mg, 0.75 mmol, 0.1 equiv.). The resulting solution wasstirred for 12 h at 90° C., and then cooled to room temperature andconcentrated under vacuum. The residue was applied onto a silica gelcolumn and eluted with ethyl acetate/petroleum ether (1:5). Thisresulted in 2.5 g (crude) of2-methyl-3-(propan-2-yl)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazoleD as a white solid: MS m/z MH⁺=301

Intermediate E

Into a 100-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen were placed2-methyl-3-(propan-2-yl)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole(2.5 g, 7.5 mmol, 1.0 equiv., 90%), 2,4-dichloro-5-fluoropyrimidine (1.7g, 9.9 mmol, 1.2 equiv.), potassium carbonate (2.3 g, 16.7 mmol, 2.0equiv.), 1,4-dioxane (40 mL), and water (10 mL). To the solution wasadded Pd(dppf)Cl₂ (680 mg, 0.83 mmol, 0.10 equiv.). The resultingsolution was stirred for 2 h at 80° C., and then cooled to roomtemperature and concentrated under vacuum. The residue was applied ontoa silica gel column and eluted with ethyl acetate/petroleum ether (1:2).This resulted in 1.2 g (48%) of5-(2-chloro-5-fluoropyrimidin-4-yl)-2-methyl-3-(propan-2-yl)-2H-indazoleE as an off-white solid: MS m/z MH⁺=305; ¹H NMR (300 MHz, CDCl₃, ppm): δ1.58 (d, 6H), 3.48-3.57 (m, 1H), 4.19 (s, 3H), 7.24 (d, 1H), 8.03-8.07(m, 1H), 8.47 (d, 1H), 8.68 (s, 1H).

Intermediate F

NaBH(OAc)₃ (14.4 g, 68.0 mmol, 1.1 equiv.) was added portionwise over 30min to a solution of N-ethyl piperazine (7.7 g, 67.5 mmol, 1.1 equiv.)and 6-bromopyridine-3-carbaldehyde (11.6 g, 62.5 mmol, 1.0 equiv.) in150 mL of methylene chloride. The reaction mixture was stirred for 48 h,then diluted with CH₂Cl₂ and excess 2N NaOH (aq.). The layers wereseparated, and the aqueous phase was extracted with CH₂Cl₂. The CH₂Cl₂extracts were combined, dried (Na₂SO₄), and concentrated to yield1-(6-bromo-pyridin-3-ylmethyl)-4-ethyl-piperazine F as an oil: MS m/zMH⁺=285

Intermediate G

Lithium bis(trimethylsilyl)amide (LiHMDS) (1M solution in THF, 12.7 mL,12.7 mmol, 1.2 equiv) was added to a solution of intermediate F (3.0 g,10.6 mmol, 1.0 equiv.), dicyclohexylphosphinobiphenyl (0.227 g, 0.64mmol, 0.06 equiv.), and Pd₂(dba)₃ (0.291 g, 0.32 mmol, 0.03 equiv.) in15 mL of THF. The mixture was heated at 50° C. for 3 h, then cooled toroom temperature, and filtered through a Dicalite® filtering medium. Thefiltrate was concentrated and the residue was taken up in CH₂Cl₂ andextracted twice with 10% HCl (aq.).

The HCl extracts were combined and washed with EtOAc, and the aqueousphase was basified with 1.0 N NaOH, and then extracted four times withCH₂Cl₂. The organic extracts were combined, dried over Na₂SO₄ andconcentrated to yield 1.2 g of5-(4-ethyl-piperazinyl-1-ylmethyl)-pyridin-2-ylamine G as a tan solid:MS m/z MH⁺=221.

Compound 1

A mixture of5-(2-chloro-5-fluoropyrimidin-4-yl)-2-methyl-3-(propan-2-yl)-2H-indazole(E) (700 mg 2.3 mmol, 1.3 equiv.),5-((4-ethylpiperazin-1-yl)methylpyridin-2-amine (G) (389 mg, 1.8 mmol,1.0 equiv.), Cs₂CO₃ (2.3 g, 7.2 mmol, 4.0 equiv.), Pd₂(dba)₃ (0.164 g,0.18 mmol, 0.1 equiv.) and Xantphos (0.104 g, 0.18 mmol, 0.1 equiv.) in10 mL of 1,4-dioxane was degassed with N₂, then heated while stirring at110° C. for 4 h. The mixture was cooled to room temperature, thenfiltered through a Dicalite® filtering medium, and the filter pad waswashed thoroughly with CH₂Cl₂. The filtrate was concentrated, and theresidue was purified by column chromatography on silica gel using aCH₂Cl₂-2% NH₃/MeOH gradient to afford 0.475 g ofN-(5-((4-ethylpiperazin-yl)methyl)pyridin-2-yl-5-fluoro-4-(3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-amine(1). MS m/z MH⁺489; ¹H NMR (300 MHz, CDCl₃, ppm): δ 0.97 (t, 3H, J=7.1Hz), 1.51 (d, 2H, J=7.0 Hz), 2.27-2.51 (overlapping m, 10H), 3.33 (s,3H), 3.54-3.68 (m, 1H), 4.15 (s, 3H), 7.64-8.71 (overlapping m, 7H),10.00 (s, 1H).

Compounds 1 and other selected examples (Compounds 2-31) of the presentinvention are listed in Table 1, all of which are or can be preparedaccording to the methods described above.

TABLE 1 Selected examples of the compounds Exam- ple Structure Name 1

N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoro-4-(3-isopropyl-2- methyl-2H-indazol-5- yl)pyrimidin-2-amine 2

N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoro-4-(7-fluoro-3- isopropyl-2-methyl-2H- indazol-5-yl)pyrimidin-2-amine 3

N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-4-(7-fluoro-3-isopropyl-2- methyl-2H-indazol-5- yl)pyrimidin-2-amine 4

4-(3-cyclopentyl-2-methyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 5

4-(3-cyclopentyl-7-fluoro- 2-methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 6

4-(3-cyclopentyl-7-fluoro- 2-methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 7

4-(3-cyclopropyl-2-methyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 8

4-(3-cyclopropyl-7-fluoro- 2-methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 9

4-(3-cyclohexyl-2-methyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 10

4-(3-cyclohexyl-7-fluoro-2- methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 11

5-fluoro-4-(3-isopropyl-2- methyl-2H-indazol-5-yl)- N-(5-((4-isopropylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 12

5-fluoro-4-(7-fluoro-3- isopropyl-2-methyl-2H- indazol-5-yl)-N-(5-((4-isopropylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 13

4-(3-cyclopentyl-2-methyl- 2H-indazol-5-yl)-5-fluoro- N-(5-((4-isopropylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 14

4-(3-cyclopentyl-7-fluoro- 2-methyl-2H-indazol-5- yl)-5-fluoro-N-(5-((4-isopropylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 15

5-fluoro-4-(3-isopropyl-2- methyl-2H-indazol-5-yl)-N-(5-((4-propylpiperazin- 1-yl)methyl)pyridin-2- yl)pyrimidin-2-amine 16

5-fluoro-4-(7-fluoro-3- isopropyl-2-methyl-2H- indazol-5-yl)-N-(5-((4-propylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 17

4-(3-cyclopentyl-2-methyl- 2H-indazol-5-yl)-5-fluoro-N-(5-((4-propylpiperazin- 1-yl)methyl)pyridin-2- yl)pyrimidin-2-amine 18

4-(3-cyclopentyl-7-fluoro- 2-methyl-2H-indazol-5- yl)-5-fluoro-N-(5-((4-propylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 19

4-(3-ethyl-2-methyl-2H- indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 20

4-(3-ethyl-7-fluoro-2- methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 21

4-(3-(sec-butyl)-2-methyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 22

4-(3-(sec-butyl)-7-fluoro- 2-methyl-2H-indazol-5- yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 23

4-(2-ethyl-3-isopropyl-2H- indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 24

4-(2-ethyl-7-fluoro-3- isopropyl-2H-indazol-5- yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 25

4-(3-cyclopropyl-2-ethyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 26

4-(3-cyclopropyl-2-ethyl-7- fluoro-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 27

4-(3-(cyclopropylmethyl)-2- methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine 28

4-(3-(cyclopropylmethyl)-7- fluoro-2-methyl-2H-indazol- 5-yl)-N-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 29

4-(3-cyclopropyl-2-ethyl-7- fluoro-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2- yl)pyrimidin-2-amine 30

4-(3-(sec-butyl)-2-methyl- 2H-indazol-5-yl)-N-(5-((4- ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5- fluoropyrimidin-2-amine 31

4-(3-(sec-butyl)-7-fluoro-2- methyl-2H-indazol-5-yl)-N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-2-amine

BIOLOGICAL ASSAYS

Compounds of the formula I are novel CDK4/6 inhibitors that have been orcan be evaluated for their activity according to the proceduresdescribed below.

Biochemical Assay

Cyclin D1 was added to freshly prepared reaction buffer [20 mM Hepes (pH7.5), 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij 35, 0.02 mg/mL BSA, 0.1 mMNa₃VO₄, 2 mM DTT, 1% DMSO]. CDK4 or CDK6 was delivered to the substratesolution and gently mixed. Compounds were tested in a 10-dose IC₅₀ modewith 3-fold dilution starting at 10 μM. The compounds, diluted in DMSO,were added into the kinase reaction mixture by Acoustic technology(Echo550; nanoliter range) and incubated for 20 minutes at roomtemperature. 33P-ATP (1 μM) was added to the reaction mixture toinitiate the reaction. The kinase reaction was incubated for 2 hours atroom temperature. The reactions were spotted onto P81 ion exchange paperand the kinase activity detected by filter-binding method. Curves werefitted to a nonlinear regression curve using a four parameter logisticequation (GraphPad Prism). Under these conditions, an IC₅₀ value of <1.0nM was determined for 1 in both CDK4 and CDK6 assays.

Cell Assay

The MCF7 human tumor cells were seeded in a clear polystyrene 96-wellmicroculture plate (Corning® Costar® 96-well flat bottom plate,Cat.#3997) in a total volume of 90 μL/well. After 24 hours of incubationin a humidified incubator at 37° C. with 5% CO₂ and 95% air, 10 μL of10×, serially diluted 1 in growth medium was added to each well induplicate (10 point dose response). After 72 hours of culture in ahumidified incubator at 37° C., in an atmosphere of 5% CO₂ and 95% air,the plated cells and Cell Titer-Glo® (Promega G7571) reagents werebrought to room temperature to equilibrate for 30 minutes. 100 μL ofCell Titer-Glo® reagent was added to each well. The plate was shaken fortwo minutes and then left to equilibrate for ten minutes. Themedium/Cell Titer Glo® reagent was transferred to a white polystyrene96-well microculture plate (Corning® Costar® 96-well flat bottom plate,Cat.#3917) before reading luminescence on the Tecan GENios microplatereader. Percent inhibition of cell growth was calculated relative tountreated control wells. All tests were performed in duplicate at eachconcentration level. The IC₅₀ value for the test agents was estimatedusing Prism 6.05 by curve-fitting the data using the following fourparameter-logistic equation:

$Y = {\frac{{Top} - {Bottom}}{1 + ( {X/{IC}_{50}} )^{n}} + {Bottom}}$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve. Under these conditions,an IC₅₀ value of <2 μM was determined for 1.

Table 2 summarizes biochemical and MCF7 cell-based data for the threereported CDK4/6 inhibitors 32, 33 and 34. The data was obtained underassay conditions described herein.

TABLE 2 Biochemical and cell data for known inhibitors of CDK4/6

MCF7 cells 10 <2 10 CDK4 <0.005 <0.001 <0.001 biochemical assay CDK6<0.002 <0.001 <0.002 biochemical assay

It will be understood by those of skill in the art that numerous andvarious modifications can be made to the compounds, compositions, and/ormethods of the present invention without departing from the spirit ofthe invention. Therefore, the various embodiments of the presentinvention described herein are illustrative only, and are not intendedto limit the scope of the invention in any way. All references citedherein are incorporated by reference in their entirety.

What is claimed is:
 1. A compound of formula (Ia)

or a pharmaceutically acceptable salt thereof, wherein: R¹ is hydrogenor ethyl; R² is isopropyl; R³ is methyl; R⁴ is hydrogen or halogen; andR⁵ is hydrogen or halogen.
 2. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is ethyl; R⁴ ishydrogen or fluoro, and R⁵ is hydrogen or fluoro.
 3. The compound ofclaim 2, or a pharmaceutically acceptable salt thereof, wherein R⁴ ishydrogen and R⁵ is fluoro.
 4. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is hydrogen; R⁴ ishydrogen or fluoro, and R⁵ is hydrogen or fluoro.
 5. The compound ofclaim 4, or a pharmaceutically acceptable salt, solvate, or prodrugthereof, wherein R⁴ is hydrogen and R⁵ is fluoro.
 6. A pharmaceuticalcomposition comprising a compound of formula (Ia) according to claim 1,or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable adjuvants, diluents, and/or carriers.
 7. Thepharmaceutical composition of claim 6, wherein in the compound offormula (Ia), R⁴ is hydrogen or fluoro and R⁵ is fluoro.
 8. A method ofrelieving, ameliorating, or modulating a disease, disorder, or conditionmediated through activity of at least one cyclin-dependent kinase (CDK),the method comprising administering to a subject in need thereof atherapeutically effective amount of the compound of formula (Ia)according to claim 1, or a pharmaceutically acceptable salt orcomposition thereof.
 9. The method of claim 8, wherein the CDK is CDK4,CDK6, or a combination thereof.
 10. The method of claim 8, wherein thedisease or disorder is cancer.
 11. The method of claim 10, wherein thecancer is selected from the group consisting of colorectal cancer,breast cancer, lung cancer, especially non-small cell lung cancer(NSCLC), prostate cancer, glioblastoma, mantel cell lymphoma (MCL),chronic myeloid leukemia (CML), and acute myeloid leukemia (AML), andcomplications thereof.
 12. The method of claim 11, wherein in thecompound of formula (Ia), R¹ is ethyl, R⁴ is hydrogen, and R⁵ is fluoro.13. The method of claim 8, wherein the disease or disorder is aninflammation-related disease or condition selected from arthritis andcystic fibrosis.
 14. The method of claim 13, wherein in the compound offormula (Ia), R¹ is ethyl, R⁴ is hydrogen, and R⁵ is fluoro.
 15. Amethod of inhibiting cell proliferation comprising treatingproliferative cells with an effective amount of a compound of claim 1,or a salt or composition thereof.
 16. A method of inhibiting acyclin-dependent kinase (CDK) selected from the group consisting ofCDK4, CDK6, and a combination thereof, the method comprising treatingthe kinase with an effective amount of a compound of claim 1, or a saltor composition thereof.
 17. A method of preparing a compound of formula(Ia), comprising a step of coupling intermediate E with intermediate G:

wherein R¹ is ethyl, R² is isopropyl, R³ is methyl, R⁴ is hydrogen orfluoro, and R⁵ is hydrogen or fluoro, and X³ is Cl, Br, or I.
 18. Themethod of claim 17, wherein the intermediate E is prepared by convertingintermediate C to intermediate D and coupling the intermediate D with apyrimidine compound H:

wherein R¹ is ethyl, R² is isopropyl, R³ is methyl, R⁴ is hydrogen orfluoro, and R⁵ is hydrogen or fluoro; and wherein R^(x) and R^(y) areindependent alkyl, cycloalkyl, or alternatively together form analkylene group, each optionally substituted by one or more substituentsindependently selected from C₁-C₄ alkyl.
 19. The method of claim 17,wherein the intermediate C is prepared by converting starting materialS1 to intermediate A and converting the intermediate A to theintermediate C:

wherein X is Cl, Br, I, or MeSO₃—; R² is isopropyl, and R³ is methyl; oralternatively the intermediate C is prepared by converting theintermediate A to an alcohol intermediate B followed by reduction of thealcohol intermediate B to form the intermediate C:

wherein R² is isopropyl, R³ is methyl, and R⁴ is hydrogen or fluoro. 20.The method of claim 17, wherein the intermediate G is prepared throughcoupling the pyridine aldehyde compound S2 and the piperazine compoundS3 to form an intermediate F, followed by converting the intermediate Fto the intermediate G:

wherein X⁴ is Br or I; and R¹ is ethyl.